RESUMEN
A cavity was made in the brain (entorhinal cortex) of developing or adult rats, and a small piece of Gelfoam was emplaced to collect fluid secreted into the wound. The neuronotrophic activity of the fluid was assayed with sympathetic and parasympathetic neurons in culture. The results show that wounds in the brain of developing or adult rats stimulate the accumulation of neuronotrophic factors and that the activity of these factors increases over the first few days after infliction of the damage.
Asunto(s)
Lesiones Encefálicas/fisiopatología , Encéfalo/fisiología , Factores de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa , Fibras Adrenérgicas/fisiología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Fibras Colinérgicas/fisiología , Cinética , Factores de Crecimiento Nervioso/farmacología , Ratas , Ratas Endogámicas , Cicatrización de HeridasRESUMEN
Neuronotrophic factors, a class of macromolecules thought to be present within the neuronal environment are required to support the survival in vitro of peripheral neurons. In the present study we have established bioassay culture systems suitable for the identification of similar agents for intrinsic neurons of the central nervous system. The striatum, hippocampus and septum of 18 day fetal rats were dissociated and plated in a serum-free medium on a neurite conducive substratum which allows an easy recognition of neurons under phase contrast microscopy. These cultures contain predominantly neurons as assessed by tetanus toxin labelling, a well recognized neuronal marker. Seeding the cell suspensions at decreasing densities yields after 24 h a density dependent survival of the neuronal population. Thus a low seeding density could be chosen where survival of these neurons required an exogenous source of trophic factors. Survival of central neurons was promoted by several conditioned media derived from rodent glial cell cultures, both primary (astroglia, Schwann) and clonal (C6 glioma, Schwannoma). Serial dilutions of these media allowed the titration of their respective neuronotrophic activities. In addition, conditioned media derived from the central neuronal cultures themselves, when seeded at a high density, were also able to support the survival of low density seeded central neurons.
Asunto(s)
Sistema Nervioso Central/análisis , Proteínas del Tejido Nervioso/análisis , Animales , Recuento de Células , Supervivencia Celular , Células Cultivadas , Sistema Nervioso Central/citología , Medios de Cultivo , Técnicas Citológicas , Feto , Métodos , Factores de Crecimiento Nervioso , Neuroglía , Neuronas/análisis , Ratas/embriología , Ratas EndogámicasRESUMEN
The hippocampus taken from E18-E19 rat embryos was dissociated into a cell suspension and was either grafted into the hippocampus of adult rats or cultured. The growth of GABAergic neurons was examined using a GABA directed antiserum. The implanted tissue was capable of survival and growth without exhibiting a laminar organization. Most of the various morphological neuronal types could be observed, establishing different types of synapses; however, granule neurons were rarely encountered. A substantial proportion of GABA-positive neurons was detected within the graft with profuse labelling of the neuropil. In cultures issued from the same cell suspension, GABA-immunoreactive neurons were numerous and had different morphologies. Altogether these data suggest that GABA neurons express a high potential for growth and sprouting in vitro and in vivo.
Asunto(s)
Hipocampo/citología , Neuronas/trasplante , Ácido gamma-Aminobutírico/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Supervivencia de Injerto , Hipocampo/embriología , Hipocampo/ultraestructura , Masculino , Microscopía Electrónica , Neuronas/fisiología , Neuronas/ultraestructura , Ratas , Ratas EndogámicasRESUMEN
Protein kinase C (PKC) is present in high concentrations in neuronal tissues and participates in various neuronal functions. Ten isoforms have so far been identified and each PKC isoform may be activated by a variety of stimuli. By immunoblot analysis the presence of PKC isoforms was examined in dissociated cell cultures of the hippocampus and during the development in vivo. As soon as embryonic day 17 the hippocampus contains detectable amounts of PKC epsilon and zeta and low levels of PKC alpha. PKC beta and gamma appear during the first and second post-natal week. All isoforms progressively increase until the adult age. Cultures of hippocampal neurons derived from rat embryos express PKC alpha, epsilon and zeta whereas PKC beta and gamma are undetectable; a distinct pattern is observed in cultures of hippocampal glial cells. The neuronal levels of PKC alpha, epsilon and zeta increase during the period in culture and are enhanced when hippocampal neurons are exposed to the continuous presence of basic fibroblast growth factor. Immunofluorescence of PKC epsilon and zeta occupies all the cytoplasmic neuronal compartment. The early expression of some PKC isoforms in cultures of post-mitotic hippocampal neurons suggest their involvement in morphological events that occur during this period; in particular the neuritic outgrowth.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/farmacología , Hipocampo/enzimología , Isoenzimas/metabolismo , Neuronas/enzimología , Proteína Quinasa C/metabolismo , Animales , Células Cultivadas , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/embriología , Técnicas de Inmunoadsorción , Neuronas/efectos de los fármacos , Embarazo , RatasRESUMEN
Subcellular fractions prepared from the rat hippocampus, were assessed for the presence of trophic activities. The cytosol of synaptosomal fractions induced mitotic reinitiation of confluent 3T3 fibroblasts. The synaptosomal fraction, enriched in mossy fibre terminals, contained the highest mitotic activity. The mitogenic activity was heat and trypsin sensitive, suggesting that polypeptides are involved. The cytosol of the mossy fibre synaptosomal fraction promoted neuritic outgrowth of PC 12 cells and embryonic hippocampal neurones in primary cultures. These results suggest that mossy fibres contain both mitogenic and neurotrophic activities. These factors could participate in mossy fibre sprouting that occur following brief seizures or experimental lesions.
Asunto(s)
Axones/fisiología , Hipocampo/fisiología , Factores de Crecimiento Nervioso/aislamiento & purificación , Sinaptosomas/fisiología , Animales , Axones/química , División Celular/efectos de los fármacos , Células Cultivadas , Citosol/química , Hipocampo/química , Hipocampo/embriología , Masculino , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Células PC12/efectos de los fármacos , Ratas , Ratas Wistar , Sinaptosomas/químicaAsunto(s)
Histamina/metabolismo , Neurotransmisores/metabolismo , Animales , Tronco Encefálico/metabolismo , Cerebelo/metabolismo , Corteza Cerebral/enzimología , Corteza Cerebral/metabolismo , Cuerpo Estriado/metabolismo , Diencéfalo/fisiología , Histidina Descarboxilasa/metabolismo , Hipotálamo Anterior/metabolismo , Masculino , Norepinefrina/metabolismo , Ratas , Serotonina/metabolismoRESUMEN
Isolated neurons from mouse neonate dorsal root ganglia were analyzed using both whole-cell clamp and single-channel recording techniques and presented a complex repertoire of potassium (K) channels. Different types of potassium channels have been found: calcium-activated K channel presenting a large unit conductance of 260 pS in symmetrical K; voltage-dependent K channels of 130 pS without calcium-dependence; two types of inward rectifying K channels (90 and 120 pS in symmetrical K); low probability K channels; delayed rectifier channels and non-selective cationic channels.
Asunto(s)
Ganglios Espinales/metabolismo , Canales Iónicos/metabolismo , Potasio/metabolismo , Animales , Animales Recién Nacidos , Calcio/metabolismo , Cationes , Conductividad Eléctrica , RatonesRESUMEN
The effects of lesions placed in the lateral hypothalamic area, i.e., interrupting the MFB (as evidenced by a 65% decrease in cortical noradrenaline and serotonin) suggest a pluricompartmentation of brain histamine (HA). The existence of an ascending histaminergic system is indicated by the reductions in l-histidine decarboxylase (H.D.) activity, in [3H]histamine synthesis and in HA content, in the cortex of lesioned rats. Moreover, the decrease in H.D. activity was restricted to the regions rostral to the lesion, without modification caudally. The time-course of the alterations in H.D. activity and in HA content is compatible with a process of anterograde degeneration. In subcellular fractionation studies, the reduction in cortical HA content was found to be mainly confined to the P2 fraction, which contains the synaptosomes. Although the widespread ipsilateral distribution of HA synthesizing terminals resembles that of monoaminergic ones, the absence of reduction in H.D. activity after selective destruction of catecholaminergic and serotoninergic neurons, by 6-hydroxydopamine or 5,6-dihydroxytryptamine respectively, renders likely the existence of specific HA-containing neurons. That the release of the amine from these neurons might be related to the nerve impulse flow is suggested by the transient effects of the lesions which preceded the degenerative process (elevated endogenous HA level and slowed rate of [3H]HA synthesis). In addition, the discrepancy between the reduction in H.D. activity and in HA level after the lesions could be explained by the presence of the amine in another neuronal system and/or in non-neuronal cells, not affected by the lesion. This additional compartment is characterized by a high HA content and a low H.D. activity and could therefore be localized in mast-cells. The respective sizes of these two compartments, estimated by several methods, appear to be approximately the same.
Asunto(s)
Histamina/metabolismo , Hipotálamo/metabolismo , Telencéfalo/metabolismo , 5-Hidroxitriptófano/metabolismo , Animales , Mapeo Encefálico , Corteza Cerebral/metabolismo , Dopa-Decarboxilasa/metabolismo , Histamina/biosíntesis , Histidina Descarboxilasa/metabolismo , Masculino , Vías Nerviosas , Norepinefrina/metabolismo , Ratas , Núcleos Septales/metabolismo , Serotonina/metabolismo , Telencéfalo/enzimologíaRESUMEN
The in vitro culture approach was combined with the cell suspension grafting technique to examine whether the maturation of dopamine (DA) neurones in vitro imposed similar limitations on their ability to survive grafting as when they are allowed to develop in situ in the fetus. The functional capacity, survival and growth of DA neurones from 2.5- and 7-day-old cultures, grafted to rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway, was compared with similar grafts freshly prepared from fetal donors of embryonic days 14, 16 and 20. Grafts of freshly dissociated mesencephalic DA neurones, taken from embryonic day 14-16 donors and 2.5-day-old cultures, generally survived well and markedly reduced amphetamine-induced rotational asymmetry in the recipient rats. However, when cultured for 7 days prior to grafting, or when taken from 20-day-old fetuses, the mesencephalic DA neurones survived very poorly and the grafts did not have any functional effects. Plating of aliquots of cell suspension used for grafting indicated that the survival rate of dissociated DA neurones is in the same order of magnitude when grown in vitro (about 2 DA neurones per 1000 cells) as when grafted in vivo to the rat striatum (about 1-5 DA neurones per 1000 cells). When the number of surviving grafted DA neurones was plotted against the behavioural effects of the grafts, a threshold number of around 100-200 DA neurones was found necessary to obtain a marked reduction (greater than 50%) in amphetamine-induced rotational asymmetry. Moreover, the survival of 300-500 DA neurones seemed to produce a 'ceiling effect' beyond which additional surviving DA neurones gave rise to little or no further effect on the amphetamine-induced rotational behaviour.
Asunto(s)
Dopamina/análisis , Mesencéfalo/citología , Neuronas/fisiología , Animales , Supervivencia Celular , Células Cultivadas , Femenino , Neuronas/análisis , Neuronas/trasplante , Ratas , Ratas EndogámicasRESUMEN
The regional distribution of histidine decarboxylase (HD) activity has been studied in the amygdaloid complex and the bed nucleus of the stria terminalis (BST) of the rat. The central and medial nuclei of the amygdala had 2-fold higher HD activity levels than the remaining nuclei of the complex. HD activity was exceptionally high in the BST, particularly in its ventral part. A lesion of the stria terminalis had no effect on this distribution whereas a combined lesion of the stria terminalis and the so-called ventral pathway induced a decrease of approximately 60% in all the amygdaloid nuclei, but not in the BST. On the other hand, a lesion of the medial forebrain bundle (MFB) induced a similar decrease in both the amygdaloid nuclei and the BST. These results confirm that HD-containing fibres are present in the MFB. On the one hand these project massively to the BST and on the other penetrate in the amygdala ventromedially along the ansa peduncularis and preferentially innervate the more medially located nuclei.
Asunto(s)
Amígdala del Cerebelo/enzimología , Carboxiliasas/análisis , Histidina Descarboxilasa/análisis , Núcleos Talámicos/enzimología , Vías Aferentes/enzimología , Animales , Masculino , Haz Prosencefálico Medial/enzimología , RatasRESUMEN
We have analyzed the brain pattern and time-course of c-fos-like proteins expression in kainic acid-induced seizures in the rat. C-fos-like immunoreactivity increased initially in the hippocampus, notably in the dentate gyrus, at the time of the first limbic motor seizure (90 min after kainate). C-fos-like labelling progressively involved different structures of the limbic system when the rats manifested a permanent epileptic state (3-6 h). The labelling was still conspicuous 12 h after kainate treatment and progressively declined to reach control levels 48 h after kainate. This time-course is similar to that produced by kainic acid on 2-deoxyglucose consumption and correlates with the electrographic changes previously described, supporting the idea that c-fos-like immunostaining may provide a useful marker of neuronal activity, with a cellular resolution. Since anoxic-ischemic treatment produces a very slight and transient increase in c-fos-like immunostaining restricted to the fascia dentata, c-fos-like expression is seizure-related and not due to a local hypoxia or ischemia.
Asunto(s)
Química Encefálica/efectos de los fármacos , Ataque Isquémico Transitorio/metabolismo , Ácido Kaínico/farmacología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Convulsiones/metabolismo , Secuencia de Aminoácidos , Animales , Conducta Animal/efectos de los fármacos , Técnicas para Inmunoenzimas , Ataque Isquémico Transitorio/fisiopatología , Masculino , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-fos , Ratas , Ratas Endogámicas , Convulsiones/inducido químicamente , Convulsiones/fisiopatologíaRESUMEN
Electrolytic lesions of the medial forebrain bundle induce a fall in histidine decarboxylase activity (the specific synthetic enzyme of brain histamine) in the ipsilateral cerebral cortex and hippocampus of the guinea pig brain; these results suggest the presence of an ascending histaminergic pathway in the guinea pig brain similar to that described in the rat. Possible alterations in the sensitivity of histaminergic receptors present in the target areas were studied following this type of lesion by combining electrophysiological and biochemical approaches. Microiontophoretic applications of histamine or noradrenaline reveal a hypersensitivity (lower ejecting currents for threshold and maximal responses) in cortical neurons ipsilateral but not contralateral to the lesion, whereas responses to iontophoretically applied GABA are not modified. In contrast the responsiveness of histamine-sensitive cyclic AMP generating systems is not modified, neither in the cerebral cortex nor in the hippocampus after this type of lesion. Similar conclusions are reached from the data obtained with specific agonists of the two classes of histaminergic receptors and measurements in the presence of a phosphodiesterase inhibitor. Several hypotheses are discussed in order to reconcile the finding of a denervation hypersensitivity revealed by iontophoresis contrasting with an unaltered responsiveness of the histaminergic receptors linked to the adenylate cyclase.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Hipocampo/efectos de los fármacos , Histamina/farmacología , Receptores Histamínicos/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Corteza Cerebral/enzimología , AMP Cíclico/metabolismo , Desnervación , Dopa-Decarboxilasa/metabolismo , Cobayas , Hipocampo/enzimología , Histamina/metabolismo , Histidina Descarboxilasa/metabolismo , Masculino , Haz Prosencefálico Medial/fisiología , Neuronas/efectos de los fármacos , Norepinefrina/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Neuronotrophic activity accumulates in a wound cavity created in the entorhinal/occipital cortex of developing rats. These trophic factors support the survival of neurons in monolayer cultures of chick embryo spinal cord, ciliary ganglion, sympathetic ganglion and dorsal root ganglion, as well as of mouse dorsal root ganglion. Trophic activity was very low both in non-injured brain tissue and in the wound cavity 1 day post-lesion, but it increased 15- to 300-fold during the subsequent 2-5 days. Together with the trophic activity in the wound fluid were other substances which interfered with the survival of spinal cord neurons. The neuronotrophic factors appeared to be proteins immunologically distinct from mouse submaxillary nerve growth factor. Fragments of rat embryo corpus striatum placed in the cortical wound cavity immediately after its formation showed very poor subsequent survival and no innervation of the host hippocampus. However, if implantation was delayed by 3 or 6 days with respect to the time at which the receiving cavity was made, the survival was greatly improved and innervation of the host took place. The time course for the accumulation of the trophic factors in the cavity paralleled the delay leading to increased survival of brain grafts. It is suggested that the neuronotrophic activity accumulating in the wound cavity during the delay period may be responsible for the increased survival of the implants.
Asunto(s)
Lesiones Encefálicas/fisiopatología , Neuronas/fisiología , Animales , Animales Recién Nacidos , Supervivencia Celular , Embrión de Pollo , Ganglios/fisiología , Ganglios/trasplante , Ganglios Espinales/fisiología , Ganglios Simpáticos/fisiología , Ratones , Neuronas/trasplante , Ratas , Ratas Endogámicas , Médula Espinal/fisiología , Factores de TiempoRESUMEN
Ciliary neuronotrophic factors (CNTFs) are proteins defined by their ability to promote the survival in tissue culture of chick embryo ciliary ganglionic neurons. CNTF activity has been reported in several source materials. Among these sources, the highest specific activity, i.e. 16,000 TU/mg, has been found in the intraocular tissue innervated by the ciliary ganglionic neurons. We now report that extracts of adult rat peripheral nerve and spinal nerve roots contain CNTF at a specific activity equal to or greater than the intraocular tissue. Equally high concentrations of CNTF activity are found in both motor and sensory nerves. The possible cellular source(s) of this mammalian CNTF is discussed.
RESUMEN
The effects of the hypoglycemic sulphonylurea tolbutamide, a marker of K(+)-ATP channels, on the N-methyl-D-aspartate- (NMDA) and kainate-activated currents were studied in rat hippocampal neurons in culture, using the patch-clamp technique in a whole-cell configuration. Tolbutamide (500 microM) reversibly increased the peak amplitude and the steady state level of NMDA- but not kainate-evoked currents. This effect was not glycine dependent as it was observed at low and saturated concentrations of glycine. The affinity of the NMDA receptor-channel complex for glycine did not change in the presence of tolbutamide. The action of tolbutamide on the NMDA-activated current was not mediated by K(+)-ATP channels since CsCl was added intracellularly at concentrations which completely blocked all K+ channels. Possible mechanisms explaining the effect of tolbutamide via the modulation of intracellular messengers are discussed.
Asunto(s)
Hipocampo/efectos de los fármacos , Ácido Kaínico/farmacología , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Tolbutamida/farmacología , Animales , Sitios de Unión , Células Cultivadas , Electrofisiología , Glicina/metabolismo , Glicina/farmacología , Hipocampo/citología , N-Metilaspartato/metabolismo , Neuronas/citología , Neuronas/fisiología , Ratas , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
The mossy fiber synaptogenesis has been studied in hippocampal slice cultures. In vivo mossy fiber terminals contact the thorny excrescences of CA3 pyramidal neurons over a restricted portion, i.e. the proximal part of the apical dendrite. In organotypic cultures mossy fibers expand their terminal field and invade the infrapyramidal area of the CA3 region and the supragranular layer of the dentate gyrus. Newly formed mossy fiber synapses in CA3 region were examined, through electron microscopy, in cultures taken at various time intervals. The main events of the formation of newly formed mossy fiber synapses can be summarized as follows. During the first week following explantation mossy fiber axons contact the dendritic shaft of the pyramidal dendrite and establish both symmetrical and asymmetrical contacts. Subsequent modifications occur in the postsynaptic portion facing the mossy fiber bouton: (i) a massive accumulation of polyribosomes and coated vesicles in the subsynaptic cytoplasm; (ii) undulations of the plasma membrane; (iii) disappearance of neurotubules at postsynaptic sites and appearance of a fine network of filamentous material. Later on in culture, complex giant spines invaginate within the synaptic bouton. In conclusion this study shows that CA3 pyramidal neurons following deafferentation retain the capacity to form thorny excrescences, when contacted by mossy fibers. Moreover these results suggest a crucial role for mossy fibers to induce the formation of thorny excrescences in an heterotopic localization, i.e. over the basilar dendrites of CA3 pyramidal neurons.
Asunto(s)
Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Fibras Nerviosas/fisiología , Sinapsis/fisiología , Animales , Membrana Celular/ultraestructura , Técnicas In Vitro , Microscopía Electrónica , Fibras Nerviosas/ultraestructura , Ratas , Ratas Wistar , Sinapsis/ultraestructuraRESUMEN
Whereas GABA is a major inhibitory neurotransmitter in the adult central nervous system, recent experiments performed in our laboratory have shown that the activation of GABAA receptors in the hippocampus leads to excitatory effects during the early post-natal period. The possible consequence of a depolarizing effect of GABA was assessed on the neuritic outgrowth of embryonic hippocampal neurons in culture. No morphological alterations were observed when hippocampal neurons were cultured for three days in the presence of muscimol, a GABAA receptor agonist. In contrast, the neuritic outgrowth of cultured hippocampal neurons was profoundly affected by the presence of bicuculline in the culture medium. In the presence of this GABAA receptor antagonist neurons displayed a reduction in the number of primary neurites and branching points, resulting in a concomitant decrease of the total neuritic length. Thus, this study suggests that GABA, acting on GABAA subtype of receptors, is able to affect the development of the hippocampus.
Asunto(s)
Hipocampo/citología , Neuronas/efectos de los fármacos , Receptores de GABA-A/fisiología , Ácido gamma-Aminobutírico/farmacología , Animales , Bicuculina/farmacología , Tamaño de la Célula , Células Cultivadas , Antagonistas del GABA , Muscimol/farmacología , Neuritas/ultraestructura , Neuronas/ultraestructura , Ratas , Ratas Wistar , Receptores de GABA-A/clasificación , Receptores de GABA-A/efectos de los fármacosRESUMEN
Fetal rat dopamine (DA) neurons were cultured in vitro for a 6-day period and transported, after redissociation, for 2 days prior to being grafted to the neostriatum of adult rats with 6-hydroxydopamine lesions of the ascending nigrostriatal pathway. In 2 of the 5 graft recipients that were tested for amphetamine-induced motor asymmetry, the grafts eliminated the lesion-induced turning behaviour within 3-6 weeks after transplantation. Fluorescence histochemistry revealed surviving grafts in all 6 recipients at 7 weeks after transplantation, containing between 42 and 125 DA neurons. The number of surviving DA neurons in the 3 non-compensated rats was below the minimum number of cells previously found to be necessary for functional effects on turning behaviour to occur.
Asunto(s)
Cuerpo Estriado , Dopamina/metabolismo , Mesencéfalo/trasplante , Sustancia Negra , Animales , Encefalopatías/inducido químicamente , Encefalopatías/terapia , Células Cultivadas , Femenino , Feto , Hidroxidopaminas , Vías Nerviosas , Oxidopamina , Ratas , Ratas EndogámicasRESUMEN
The prevalence of urinary antibody-coated bacteria (ACB), suggesting renal bacteriuria, was studied in three groups of patients: inpatients whose physicians suspected urinary tract infections, asymptomatic outpatients with neurogenic bladders but without urethral catheters, and asymptomatic inpatients with indwelling urethral catheters. The prevalence of ACB was 67% of inpatients with positive cultures without catheters, 94% of patients with neurogenic bladders, and 36% of patients with urethral catheters. These results suggest a high prevalence of upper urinary tract involvement in patients with positive urine cultures, even if asymptomatic.